---
title: "Viral evolution and origins of SARS-CoV-2"
author: "Duncan Golicher"
date: "01/04/2021"
output:
  slidy_presentation: default
  ioslides_presentation: default
  beamer_presentation: default
link-citations: yes
bibliography: mendeley.bib
---

```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = FALSE, warning = FALSE,message = FALSE)
library(mapview)
load("rmap.rda")
```

# Text book stuff

- Field's virology 
- Well over 2000 pages per volume (2 volumes)
- Each chapter apologises for what is **not** included
- Uncertainties, known unknowns, unknown unknowns: but very few known knowns
- Most chapters focus on pathology and molecular biology
- **Ecology** of viruses remains poorly understood

# Web of science

- Search terms "virus" + "ecology"
- Search term "coronavirus"

![](figs/wos1.png) ![](figs/wos2.png)





# Quick history

- Viruses not recognised until 1880s
- Adolf Meyer working on tobacco at Wageningen identified TMV
- Clearly an infectious agent, but could not fulfill Koch's postulates as only cultivable in living cells: 
- The issue with Koch's postulate continues to muddy some debates to this day
- Beijerinck named the agent a "contagium vivum luidum"" or a contagious living liquid. 
- d’Herelle developed the plaque assay in 1917 
- First electron micrographs were taken of tobacco mosaic virus (TMV) in 1939.

# Tobacco mosaic virus

- Remains a key experimental organism 
- Plant viruses are possibly more diverse than higher animal viruses (more species of plant)
- Plant viruses have a different set of challenges (movement through an organism with cell walls)
- Plant viruses are not thought to infect animals directly (possible exceptions)

![](figs/tmv.png)

# Bacteriophages 

- Lytic (pathogneic) and lysogenic (non pathogenic) infections identified in 1900s
- Experiments with viruses resulted in the discovery of mRNA and the role of DNA
- First complete nucleotide sequence of an RNA virus genome was the bacteriophage MS2 (1976)
- Most abundant "organisms" on the planet
- Present at roughly $10^7/mL$  in coastal seawater 
- Around $10^{31}$ individual tailed phage virions on the planet
- If all the tailed phages were laid end to end, they would extend for 200 million light years into intergalactic space.

# Phage seen through electron microspope

![](figs/phage1.png)

# Insect viruses

- Abundant, ubiquitous and highly diverse
- Mammalian and avian viruses may have evolved in insects
- Insects are also vectors for both animal and plant viruses
- Can play a role in very complex life histories
- Some extremely complex symbiotic relationships

# Parasitic wasps use viruses as "venom"

![](figs/wasp.png)

# Mammalian viruses

- Experiments on viruses of animals used immortalised human cell lines and monkey kidney cells
- Polio virus was an early focal virus
- Virology allowed key insights into oncology and cancer
- Cellular receptors began to be identified and studied in depth
- Attention mainly on pathogenic viruses of humans (and some of rodents and domestic animals)
- Full diversity of mammalian viruses remains unknown

![](figs/oncology.png)

# Viral evolution

- Fundamental aspects of viral evolution remains unknown, contentious, or both
- Precise estimates of mutation rates unknown
- Deep origins of viruses still uncertain 
- Did viruses predate cellular organisms?
- "Escaped gene theory" vs "RNA first" theory
- Did the earliest replicators resemble what we now know as viruses?
- Should viruses even be classified as life?

# Evolution and phylogeny

- Viruses generally evolve at high rates, following basic and extended Darwinian principles 
- Evolutionary time frame much shorter than that of any other organism. 
- Species concept cannot be uncontentiously applied.
- True antiquity of currently circulating viruses still vague. 
- No fossil record

# Species concept as applied to viruses

- Importance of species in biology derives from their importance in systematics
- Prior to the 1980s the notion of viral "species" was rejected by most of the scientific community 
- Clearly could not apply Ernst Mayr's definition(*"species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups"*)
- Concepts of species, strains, variants and viral isolates caused confusion [@Alimpiev2019]
- 1982 International Committee on Taxonomy of Viruses(ICTV): species = "a cluster of strains from a variety of sources, or a population of strains from a particular source, which **have in common a set of stable properties** that separate **the cluster** from other **clusters of strains.**"

# Modified definitions

- Van Regenmortel: "a polythetic class of viruses (*strains*) that constitute a replicating lineage and occupy a particular ecological niche"
- Polythetic is defined as "characteristics which occur commonly in members of a group or class, **none of which is essential** for membership of that group or class."
- 2012 ICTV viral species definition based on shared ancestry. "A monophyletic **group of viruses (strains)** whose properties can be distinguished from those of other species by multiple criteria"
- Can be used to define any virus **taxon**. (I.e. not just a species)
- Current definition essentially defines virus species (clades) based **only on their genomic sequence**
- However genetic sequences are a continuum, with no naturally defined breaks

# Overlaps between SARS-CoV-1 and SARS-CoV-2

- Around 86% sequence similarity between SARS-CoV-1 and SARS-CoV-2
- However, also considerable sequence diversity **within** each clade
- "In terms of taxonomy, SARS-CoV-2 is (just) another virus in the **species** Severe acute respiratory syndrome-related coronavirus" [@Gorbalenya2020]

![](figs/novelty.png)

# Non-identical base pairs

- Overlaps in **between** and **within** clade sequence differences [@Gorbalenya2020]

![](figs/overlaps1.png)

# Phylogeny of coronaviruses

- Key questions regarding time scale of all divergences between all coronavirus taxa.

![](figs/phylogeny.png)

# Time since divergence

Knowing time since divergence key to understanding viral evolution and viral "novelty".

- Three approaches

 1. Find a good match between the phylogenetic tree of viruses and that of their hosts. Assume co-divergence (not usually possible as few viruses are narrowly host specific .. can work for DNA viruses .. not generally RNA) 
2. Find endogenous genome copies within the host: Use the substitution rate of the host (similar to 1)
3. **Measure current evolution (i.e mutations fixed during time frame of human observation)**


# RNA viruses

- Particularly difficult to produce phylogenies
- Rarely strictly host specific
- Very fast rates of mutation
- Many RNA virus populations thought to exist as **quasispecies**
- Swarms of individual viral genomes where every member is unique. 

 
# Molecular clock

- Rate of evolution has been estimated for polio (short RNA ~7500 base pairs)
- Estimated that thes is one synonymous subsitution per 100 base pairs per year [@Jorba2008]
- Non-synonymous substitutions constrained by purifying selection
- However, these measurements were on a population with low prevalence 
- Hard to generalise, as rate of evolution depends on number of infections

 

# Quasispecies

- Experimental approaches using in vitro and in vivo **laboratory models**  focus on short-term **intrahost evolutionary mechanisms** 
- May not always be relevant to natural systems.
- Comparative approach relies on the phylogenetic analysis of natural virus populations
- Consider data collected over multiple cycles of virus–host transmission. [@Geoghegan2018]

# @Geoghegan2018

![](figs/evol.png)

# Quasispecies evolution

- Mutation–selection balance in which a distribution of variant viral genomes is ordered around the fittest, or “master,” sequence. 
- Mutation rates in RNA viruses are so high that the frequency of any variant is not only a function of its own replication rate (fitness), but also of the mutation of other variants in the population derived from the original sequences
- “Mutational coupling” leads to a distribution of evolutionarily interlinked viral genomes
- Entire mutant distribution behaves as a single unit
- Natural selection acts on the mutant distribution as a whole, rather than on individual variants.
- The "quasispecies"" as a whole evolves to maximize its average fitness. 
- Has been applied to SARS-Cov-1 [@Xu2004] and SARS-CoV-2 [@Jary2020]

# Quasispecies evolution

![](figs/evol2.png)

# Quasispecies and SARS-Cov-2?

- Coronaviruses contain the largest known RNA genomes (27 to 32 kilobases cf 7kb in polio) [@Smith2013]
- RNA viruses rely primarily on low-fidelity replication by RNA- dependent RNA polymerases 
- With no proofreading and repair functions, the average mutation rate of RNA viruses is estimated to be around one mutation per genome per round of replication . 
- RNA viruses risk crossing an error threshold at which there are too many deleterious mutations (limitation on size)
- Are Covs DNA "wannabes?"
- CoVs encode a proofreading exoribonuclease 
- Distinct from the typical RNA Polymerase
- So possibly more conserved sequences than may be expected in other long sequence RNA viruses

# Zoonosis

- Most pandemics involving viruses originate in animals 
-  Novel viruses are "*driven to emerge by ecological, behavioural, or socioeconomic changes.*" [@Smith2013]
- Sometimes claimed that humans are "*coming into contact with wild animals more frequently than in the past*"
- However, most likely source of novel pathogens are **closely related species** (particularly primates)
- Most human contact is with domestic animals

# Biomass 

![](figs/biomass1.png)

# Humans vs wildlife

- Livestock accounts for more biomass than all humans on earth; More than 50% greater than humans.
- Livestock accounts for much more than all wildlife (mammals + birds) by a factor of ten
- I.e  humans + livestock ~ 15 times the total wildlife biomass (cells in which viruses can replicate)
- Even so, most viral genomic **diversity** is found in wildlife. 
- Studies on viruses that affect neither livestock nor humans vanishingly small part of the literature

![](figs/biomass2.png) ![](figs/biomass3.png)

# Zoonoses

- Source of most **diversity** is wildlife
- Most likely source of **infection** is livestock
- Wildlife -> livestock <-> humans
- Livestock including farmed animals + household pets

# Co-infections and recombination

- Wildlife provides a large pool of unsequenced genetic material
- Co-infections between endemic viruses of domestic animals and "novel" (i.e. previously unknown sequences) likely source of zoonoses
- RNA viruses forming quasispecies particularly likely candidates. 
- Coronaviruses may be DNA "wannabes" however they form subgenomic RNA making recombination more likely
- Recombination makes tracing evolution very hard
- Availability of sequences key to understanding "novel" recombinants
- Availability bias (do we have all the sequences?)

# Recombination in SARS?

![](figs/civet_recomb.png)
[@Graham2010]

# Bats, cats, rats (+ mustelids and primates)

-  ~ 2000 species placed of rodents: 40% of mammals
-  ~ 925 species of bats: 20% 
-  ~ 230 species of primates
-  ~ 60 species of mustelids
-  ~ 36 species of cats


# Why bats?

- Most likely source of zoonoses would appear to be rodents
- However, bats are both **highly diverse** and highly **mobile**
- Proximity of insectivorous bats to humans goes largely unnoticed (nocturnal)

# Bat viral diversity

[@Wu2016] 

![](figs/bat_viral_diversity.png)

# Studies of bat coronavirus diversity

[@Drexler2014]

![](figs/distribution_bat_studies.png)


# Rhinolophus

```{r}
mp
```


# Illustrative studies in bats

- Multiple examples of coronavirus diversity in bats throughout the world (see reference section for titles and links)

- @Balboni2011 @Balboni2012 @Monchatre-Leroy2017 @Hu2017 @Rizzo2017 @Hayman2013 @Plowright2014 @Mendenhall2019 @Lacroix2020 @Wong2019@Anthony2013 @Tao2019 @Drexler2014a @Woo2012 @Smith2016a @Woo2006 @Lau2015 @Corman2015 @Drexler2011

# Inter genus host switches

[@Latinne2020]

![](figs/intergenus.png)

# "Spillover"

[@Cui2019]

![](figs/spillover.png)

# Cats as intermediate hosts

- Civet cats are farmed in China, but comparatively rare
- Domestic cats are ubiquitous
- Feline ACE2 reacts with SARS-Cov as well as human ACE2  [@Guo2008] [@Jo2020]

![](figs/carnivores.png)


# Cats in china

- Thousands of cats were rounded up and killed in China prior to Olympic games in Beijing

![](figs/cat_cull.png)

# Cats and SARS-Cov-2

[@Zhang2020e]



![](figs/china_cat_sero.png)

[@Musso2020]

![](figs/italian_cat.png)

# Pangolins

- Pangolin theory based on a single sequence [@Segreto2020]
- No evidence in wild populations [@Lee2020]

# Furin cleavage site

- Main objection to the zoonotic origin theory
- Furin cleavage sites are actually very common in betacoronavirus [@Wu2021]
- S1/S2 occurs in three clades: MERS-CoV strains, the bat coronavirus HKU5 strains, and coronavirus Neoromicia/PML-PHE1/RSA/2011. 
- MERS-CoV and bat coronavirus HKU5 are the only clades in Merbecovirus having furin cleavage site at S2′. In Embecovirus, furin recognition motif at spike S1/S2 is universal:
- Also universal in Gammacoronavirus, and occurs in two clades of Alphacorovanvirus **including feline coronavirus** and relatives.
- HCoV NL63 also has a furin cleavage site at spike S2′

# @Muth2018

![](figs/muth.png)

# @Corman2020

![](figs/corman1.png)

# Assumptions of lab origin theory

- Evolutionary rate known (comparatively slow and stepwise) so unlikely to lead to change
- Most possible sequences already known
- Furin cleavage site (which is absent in most bat sequences) could not have arisen "by chance"
- SARS-Cov-2 must have arisen in China
- Wuhan wet market origin implausible
- No intermediate host (as the virus was "designed in lab")
- GOF experiments were known to be taking place in Wuhan labs
- Escape from labs plausible and likely
- Accidental escape as plausible as deliberate escape

# Assumptions of zoonotic origin theory

- Evolutionary rates unknown
- Quasispecies evolution likely
- Recombination both possible and likely
- Most of the possible available sequences remain unknown
- Furin cleavage site is a common "lemma"" in many other coronviruses
- Intermediate host could be a common, domestic animal
- No single clearly defined "point of origin"

# Implications of lab origin theory

- Current situation arose as a result of nefarious intent or human negligence
- Humans are "to blame" for the pandemic
- Existing knowledge was available that could have avoided the situation arising
- Political will to prevent the pandemic was lacking
- Fund more applied research to prevent future pandemics
- Viruses are dangerous
- Apparent "design" implies a "designer" (echos of anti-Darwinism)

# Implications of the zoonotic origin theory

- Current situation was unavoidable.
- Arose through natural evolutionary processes (extended Darwinian evolution)
- Humans are not to blame for the pandemic. They are to blame for the response to it. 
- Insufficient basic knowledge was available to prevent the situation.
- Fund more basic research to fill fundamental gaps in our understanding of life.
- Viruses are a fundamental part of ecosystems. Some are bad, some are good, all are part of a complex system
- We can't live without viruses. 


# References
